藏红花素通过Nrf2/HO-1通路对脑缺血再灌注大鼠血脑屏障的保护作用

Protective effect of crocin on the blood-brain barrier of rat with cerebral ischemia reperfusion through Nrf2/HO-1 pathway

[目的]探讨藏红花素对脑缺血再灌注大鼠血脑屏障(BBB)的保护作用及其机制。[方法]按随机数字表法将240只健康SD大鼠分为假手术组,模型组,低(10 mg/kg)、中(20 mg/kg)、高(40 mg/kg)剂量藏红花素组和尼莫地平(2 mg/kg)组,每组40只;采用夹闭大脑中动脉2 h的方法复制脑缺血再灌注大鼠模型,各组分别于造模手术前7 d开始每日1次腹腔注射给药(假手术组和模型组均给予0.9%氯化钠溶液)。再灌注24 h后,进行神经功能缺失评分,红四氮唑(TTC)染色法计算脑组织梗死率,失质量法检测脑组织含水量;苏木精-伊红(HE)染色法观察脑组织病理学改变;伊文思蓝渗透法检测BBB通透性;酶联免疫吸附(ELISA)法检测脑组织肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、干扰素-γ(IFN-γ)含量;分光光度法检测丙二醛(MDA)含量和超氧化物歧化酶(SOD)、过氧化氢酶(CAT)活性;蛋白免疫印迹(Western blot)法检测脑组织闭合蛋白(Occludin)、闭锁连接蛋白-1(ZO-1)、核因子E2相关因子2(Nrf2)、血红素加氧酶-1(HO-1)、胞浆核因子-κB p65(NF-κB p65)、胞核NF-κB p65蛋白表达。[结果]与假手术组比较,模型组大鼠神经功能缺失评分、脑梗死体积率、脑含水量均升高(P<0.05);脑组织可见水肿,神经元数量减少、空泡变性,胞核偏移、固缩深染,炎性细胞浸润等病理学改变;脑组织伊文思蓝渗透量升高(P<0.05);脑组织TNF-α、IL-1β、IFN-γ、MDA含量升高,SOD、CAT活性降低(P<0.05);脑组织Occludin、ZO-1、Nrf2、HO-1表达量降低,胞浆NF-κB p65、胞核NF-κB p65表达量和胞核NF-κB p65/胞浆NF-κB p65比值升高(P<0.05)。与模型组比较,中、高剂量藏红花素组和尼莫地平组神经功能缺失评分、脑梗死体积率、脑含水量降低(P<0.05);脑组织病理学改变明显改善,伊文思蓝渗透量(P<0.05);TNF-α、IL-1β、IFN-γ、MDA含量降低且SOD、CAT活性升高(P<0.05);Occludin、ZO-1、Nrf2、HO-1表达量升高,胞浆NF-κB p65、胞核NF-κB p65表达量和胞核NF-κB p65/胞浆NF-κB p65比值降低(P<0.05)。[结论]藏红花素对脑缺血再灌注大鼠BBB结构和通透性具有一定的保护作用,能够减轻脑组织损伤,其机制可能与激活Nrf2/HO-1通路,抑制NF-κB入核,进而抑制氧化应激和炎症反应有关。

[Objective]To investigate the protective effect and mechanism of crocin on the blood-brain barrier(BBB)of rats with cerebral ischemia-reperfusion.[Methods]According to the random number table method,240 healthy SD rats were divided into sham operation group,model group,low(10 mg/kg),medium(20 mg/kg),high(40 mg/kg)dose crocin group and nimodipine(2 mg/kg)group,40 in each group.The cerebral ischemia-reperfusion rat models were replicated by clamping the middle cerebral artery for 2 hours.The 7 days before the model operation,the drugs were injected intraperitoneally once a day,and the rats in sham operation group and model group were given 0.9%sodium chloride solution.The 24 h after reperfution,the neurological deficit score was performed,the brain tissue infarction rate was calculated by red tetrazolium(TTC)staining method,the water content of brain tissue was detected by weightlessness method;the pathological changes of brain tissue was observed through HE staining;the BBB permeability was detected by evans blue permeation method;the content of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),interferon-γ(IFN-γ)were detected by ELISA method;the content of malondialdehyde(MDA)and the activity of superoxide dismutase(SOD),talase(CAT)in brain tissue were detected by spectrophotometry method;the expression of Occludin,zonula occludens-1(ZO-1),nuclear factor E2 related factor 2(Nrf2),heme oxygenase 1(HO-1),cytoplasmic nuclear factor-κB p65(cytoplasmic NF-κB p65),nucleus NF-κB p65 in brain tissue were detected by Western blot method.[Results]Compared with the sham operation group,the neurological deficit score,infarction rate,water content of the rats in model group were increased(P<0.05);the brain tissue presents pathological changes such as edema,decreased neuron number,vacuolar degeneration,nucleus deviation,pyknosis and hyperchromatism,inflammatory cell infiltration;the penetration of evans blue in brain tissue was increased(P<0.05);the content of TNF-α,IL-1β,IFN-γ,MDA in brain tissue were increased while the activity of SOD,CAT were decreased(P<0.05);the expression of Occludin,ZO-1,Nrf2,HO-1 were decreased,while the expression of cytoplasmic NF-κB p65,nuclear NF-κB p65 and the ratio of nuclear NF-κB p65/cytoplasmic NF-κB p65 were increased(P<0.05).Compared with the model group,the neurological deficit score,infarction rate,water content of the rats in medium,high dose crocin group and nimodipine group were significantly decreased(P<0.05);the pathological changes of brain tissue was improved;the penetration of evans blue in brain tissue was decreaed(P<0.05);the content of TNF-α,IL-1β,IFN-γ,MDA in brain tissue were decreased while the activity of SOD,CAT were increased(P<0.05);the expression of Occludin,ZO-1,Nrf2,HO-1 were increased,while the expression of cytoplasmic NF-κB p65,nuclear NF-κB p65 and the ratio of nuclear NF-κB p65/cytoplasmic NF-κB p65 were decreased(P<0.05).[Conclusion]Crocetin has a certain protective effect on the structure and permeability of BBB in rats with cerebral ischemia-reperfusion,and can reduce brain tissue damage;which mechanism may be related to activation of Nrf2/HO-1 pathway and inhibition of NF-κB into the nucleus,and thereby inhibiting oxidative stress and inflammation.